Expression and localization of integrin alpha51 were analyzed on human RPE by immunoblots analysis and immunofluorescence analysis. SearchLight technology was used to detect the secretion of pro-inflammatory cytokines and angiogenesis growth factors. RPE attachments to different extracellular substrates were determined using CytoMatrix screening kits. BrdU incorporation and wound-healing assays were used to test hfRPE proliferation and migration. F-actin cytoskeleton was visualized with phalloidin immunofluorescence staining. Integrin alpha51 was detected in native adult and fetal human RPE. The alpha5 subunit is predominantly localized at the apical membrane of hfRPE, while the 1-subunit is uniformly detected at the apical and basolateral membranes. We also found that hfRPE cultures secrete significant amounts of fibronectin to the apical bath. The apical localization of alpha51 and the secretion of fibronectin to the apical bath suggest the presence of an autocrine loop that can guide the migration of RPE. JSM6427 (a specific integrin alpha51 antagonist), but not JSM8009 (negative control compound) significantly inhibited hfRPE cell attachment to fibronectin, but not laminin, or collagen I or IV. JSM6427 also showed a strong inhibitory effect on bFGF, PDGF-BB or serum induced cell migration and proliferation. Furthermore, JSM6427 induced significant disruption of the F-actin cytoskeleton of dividing RPE cells but had no effect on quiescent cells. The strong inhibitory effects of JSM6427 on human RPE cell attachment, proliferation, and migration is probably mediated by F-actin cytoskeletal disruption in proliferating cells and suggests a potential clinical use of this compound in proliferative retinopathies.